Activation of G protein-gated inwardly rectifying potassium (GIRK) channels provides a key inhibitory signal in the brain that reduces neuronal firing. Many neurotransmitters in the reward pathway, such as dopamine and GABA, couple to GPCRs that signal through GIRK channels. Prior studies have established a role for GIRK channels in the response to psychostimulants. A complex network of proteins is hypothesized to support drug-dependent changes in trafficking and targeting of GIRK channels to postsynaptic inhibitory synapses, but the identity of these proteins remain largely unknown. To address this major gap in the field, an innovative technique of proximity-dependent biotin identification (referred to as iBio-ID) will be used to identify new proteins in the GIRK channel proteome (Aim 1). In this proposal, GIRK channels will be engineered to biotinylate proteins in vivo (within 50 nanometers of the channel), and then biotinylated proteins will be purified and identified using tandem mass spectrometry. Preliminary data show that GIRK channels fused to the biotinylating enzyme are functional, and can biotinylate proteins in vivo, leading to identification of putative GIRK channel regulators. Following quantitative analyses, candidate GIRK channel regulator proteins will be assessed for functional interaction with GIRK channels in neurons. Changes in the GIRK channel proteome with cocaine, using the locomotor sensitization as a model of addiction, will also be studied (Aim 2). These experiments will provide for the first time a comprehensive list of proteins in the GIRK channel proteome, and lead to the identity of new drug-dependent regulators of GIRK channels. Discovery of novel protein targets in the reward pathway could lead to the development of new treatments for addiction.